PROJECT SUMMARY The prevalence of non-alcoholic fatty liver disease (NAFLD) is almost 30% in the western world and is expected to rise in the next decade. NAFLD is characterized by protein misfolding in the endoplasmic reticulum (ER) activating the unfolded protein response (UPR). Prolonged ER stress conditions further lead to oxidative stress, protein aggregation, organelle damage, cellular bioenergetic collapse, and eventually cell death. Recent studies indicate that protein misfolding in the ER contributes to hepatocyte failure associated with NAFLD, non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). Through detailed characterization of hepatocyte function, our preliminary results lead us to propose that ER protein misfolding causes mitochondrial dysfunction that leads to further protein misfolding culminating in hepatocyte failure. Although previous studies demonstrated that protein misfolding in the ER of hepatocytes is associated with metabolic syndrome and NAFLD, there has never been a careful characterization of how ER protein misfolding causes catastrophic cellular events leading to oxidative damage, fibrosis and cell death. We have shown that misfolding of coagulation factor VIII (FVIII) in the ER disrupts complex 1 of the electron transport chain. Amazingly, we demonstrated that treatment with a mitochondrial-targeted antioxidant corrects he defective mitochondrial function and also improves FVIII folding in the ER in cell culture. This unprecedented finding indicates an unappreciated association between ER protein misfolding and defective mitochondrial bioenergetics. In the proposed studies, we will use two separate models to induce ER stress in vivo in hepatocytes; (i) excess nutrition of a high fructose diet, or (ii) expression of FVIII to elucidate how ER protein misfolding disrupts mitochondrial bioenergetics and dynamics. Presently, ongoing clinical studies are using viral delivery of FVIII to hepatocytes in hemophilia A patients. Furthermore, we will apply non-biased technologies of metabolomics and RNA-Seq using novel genetic murine models to elucidate how protein misfolding causes Ca2+ leak from the ER and entry into the mitochondrial matrix to disrupt mitochondrial bioenergetics and/or dynamics. Evidence supports that ER protein misfolding and defective mitochondrial function exist in all degenerative diseases. In addition, extensive findings demonstrate that protein misfolding in the ER is associated with liver failure in a number of common acute conditions including viral infection, ethanol toxicity, acetaminophen toxicity and ischemia reperfusion injury. Our fundamental novel exploration into unchartered territory will undoubtedly generate new hypotheses concerning the impact of ER protein misfolding on the mitochondrial electron transport chain. The studies will provide fundamental mechanistic insight into how ER and mitochondrial functions are reciprocally regulated and lead to novel therapies for NAFLD and other diseases of protein misfolding.